Glioblastoma multiforme (GBM) is a World Health Organization Grade IV cancer, the most malignant category of glial tumors with median survival time less than one year. The combined temozolomide (TMZ)-mediated chemoradiotherapy only modestly improves survival of GBM patients [2-yr survival rate of 27%] and 80% of totally resected GBM recur. The key challenge in the treatment is an increase of a subpopulation of GBM cancer cells which are resistant to apoptosis. Therefore, new strategies are needed to improve the efficiency of the current therapies for GBM. TMZ causes a DNA O6-methylguanine lesion which triggers DNA repair, depletes the enzyme O6-methylguanine methyltransferase, and leads to apoptotic cell death. The hallmark of apoptosis is a drastic reduction in cell volume resulting from loss of K+i and Cl-i, termed apoptotic volume decrease "AVD". AVD is an ubiquitous characteristic of apoptosis which is independent of the death stimuli. Loss of cell volume and reduction of total intracellular ionic strength (via loss of K+ and Cl-) occur before any other detectable characteristics of apoptosis. The reduction of intracellular ionic strength has been suggested to play a permissive role in activation of caspases and triggering the entire caspase cascade and apoptotic machinery. Normally, cells respond to volume perturbations by activating volume regulatory mechanisms. The process by which shrunken cells return to normal volume is termed regulatory volume increase (RVI). RVI can only be regulated by the gain of osmotically active solutes such as Na+, K+ and Cl-. Na+-K+-2Cl- co-transporter isoform 1 (NKCC1), which transports 1 Na+, 1 K+ and 2 Cl- ions into the cell, is the primary cell volume regulatory protein in RVI in response to either hypertonic or isotonic cell shrinkage. Therefore, NKCC1-mediated RVI will promote cell survival. However, it remains unexplored whether NKCC1-mediated K+, Cl- accumulation can counteract AVD, restore intracellular ionic strength, reduce caspase-mediated apoptosis, and promote cell survival in response to TMZ-mediated DNA damage. Our preliminary data illustrate that NKCC1 is the most important ion transport mechanism in regulating Cl-i and RVI in GBM cancer cells. Interestingly, pharmacological blockade of NKCC1 activity with its potent inhibitor bumetanide enhanced TMZ- mediated apoptosis. This led us to hypothesize that NKCC1 activity is stimulated in the TMZ-treated cells and its inhibition can sensitize glioma to TMZ-mediated apoptosis. Completion of this study will shed light on whether a combined TMZ-based therapy with NKCC1 inhibition presents a novel therapeutic strategy, which may increase the efficiency of the current chemotherapy. PUBLIC HEALTH RELEVANCE: Glioblastoma multiforme (GBM) is the most prevalent and aggressive form of primary brain tumor. The combined temozolomide (TMZ)-mediated chemoradiotherapy only modestly improves survival of GBM patients [2-yr survival rate of 27%] and 80% of totally resected GBM recur. The key challenge in the treatment is an increase of a subpopulation of GBM cancer cells which are resistant to apoptosis. Therefore, new strategies are needed to improve the efficiency of the current therapies for GBM. We hypothesize that Na-K-Cl cotransporter (NKCC1) activity is stimulated in the TMZ- treated cells and counteracts apoptotic volume decrease and apoptosis. Inhibition of NKCC1 can sensitize glioma to TMZ-mediated apoptosis. Completion of this study will shed light on whether a combined TMZ-based therapy with NKCC1 inhibition presents a novel therapeutic strategy, which may increase the efficiency of the current chemotherapy.